Abstract Colorectal cancer (CRC) is the second leading cause of cancer mortality in the USA. The failure of treatment is due to resistance to chemotherapy, one of the biggest obstacles for effective cancer therapy. Therefore, there is an urgent need to identify molecules or pathways that can be targeted to overcome drug resistance and improve cancer treatment. TGF? plays multiple functions including conferring drug resistance. However, the mechanisms underlying TGF?-mediated chemoresistance are not clear. PDK4 is one of the isoforms of pyruvate dehydrogenase kinase (PDK), which phosphorylate pyruvate dehydrogenase (PDH) and regulate glucose metabolism. We have discovered a novel function of PDK4 that mediates the response of colon cancer cells to 5-FU treatment. We show that PDK4, but not PDK 1-3, is differentially expressed in colon cancer cells, and that its expression positively correlates with the resistance to 5-FU treatment. Knockdown of PDK4 sensitizes colon cancer cells to 5-FU- or oxaliplatin-induced apoptosis. Experiments in tumor xenograft mouse models demonstrate that knockdown of PDK4 increases the effectiveness of 5-FU-mediated inhibition of tumor growth in vivo. Furthermore, for the first time, we have established a novel crosstalk between TGF? signaling and PDK4: TGF? increases PDK4 expression while PDK4 enhances TGF? signaling, which forms a positive feedback loop. Elevated PDK4 expression contributes to TGF?-mediated drug resistance in colon cancer cells. Studies of patient samples indicate that expression of PDK4 and TGF? signaling positively correlate with each other and with chemoresistance in CRC. Therefore, our studies unveil an important function of TGF?/PDK4 in mediating drug resistance in CRC. In this proposal, we will determine the mechanism(s) underlying the crosstalk between TGF? signaling and PDK4, elucidate the mechanism(s) of PDK4-mediated drug resistance and identify novel substrates of PDK4. We will determine the functional role of PDK4 in colon cancer metastasis and the contribution of PDK4 and its crosstalk with TGF? in CRC drug resistance using an orthotopic mouse model. We will also demonstrate the clinical relevance in patient samples. The completion of these studies will identify TGF?/PDK4 as a novel regulator of chemoresistance in CRC, substantially advance our understanding of molecular mechanisms underlying drug resistance and provide proof-of-concept that combinations of 5-FU with inhibitors of PDK4 or TGF? could be potentially effective therapies to overcome chemoresistance for CRC treatment.